1. Volume 9, Issue 5, Pages 1584-1593 (December 2014)
ThermoMouse: An In Vivo Model to Identify Modulators of UCP1 Expression in Brown Adipose Tissue 
Andrea Galmozzi, Si B. Sonne, Svetlana Altshuler-Keylin, Yutaka Hasegawa, Kosaku Shinoda, Ineke H.N. Luijten, Jae Won Chang, Louis Z. Sharp, Benjamin F. Cravatt, Enrique Saez, Shingo Kajimura 
Cell Reports 
Volume 9, Issue 5, Pages (December 2014)
DOI: /j.celrep
Copyright © 2014 The Authors Terms and Conditions

2. Cell Reports 2014 9, 1584-1593DOI: (10.1016/j.celrep.2014.10.066)
Copyright © 2014 The Authors Terms and Conditions

3. Figure 1 Luciferase Imaging of UCP1 Expression In Vivo
(A) 3D reconstruction of luciferase signals in Ucp1 luciferase reporter mice. Adipose depots with specific luciferase signal are indicated.
(B) Quantification of luciferase in adipose depots, skeletal muscle, and liver of mice kept at room temperature. Values normalized to protein content.
(C) UCP1 protein expression in tissue lysates from the mouse in (B).
(D) Luciferase activity in Ucp1 luciferase reporter mice kept at 28°C and subsequently kept at 9°C for 24 hr. Representative mice are shown.
(E) Quantification of luciferase signal in interscapular BAT of Ucp1 luciferase reporter mice shown in (D) (n = 9). ∗∗∗p < Data are expressed as mean ± SEM.
(F) Ucp1 mRNA expression in interscapular BAT of Ucp1 luciferase reporter mice kept at 28°C and 9°C for 24 hr. ∗∗∗p < Data are expressed as mean ± SEM.
(G) UCP1 protein expression in interscapular BAT of transgenic mice analyzed in (F).
(H) Representative image of luciferase signal in Ucp1 luciferase reporter mice treated with saline or CL-316,243 for 1 day (acute) or 4 days (subchronic; n = 3).
(I) UCP1 protein expression in BAT of transgenic mice analyzed in (H).
(J) Representative images of luciferase signal in inguinal WAT depots of Ucp1 luciferase reporter mice treated with saline or CL-316,243 for 4 days.
(K) UCP1 protein expression in inguinal WAT depots of Ucp1 luciferase mice treated with saline or CL-316,243 for 1 day or 4 days.
Cell Reports 2014 9, DOI: ( /j.celrep )
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4. Figure 2 Cell-Based System to Monitor UCP1 Expression
(A) Luciferase activity in immortalized Ucp1 luciferase brown adipocytes. Differentiated adipocytes were treated with DMSO (control) or rosiglitazone for 5 days (n = 3).
(B) UCP1 protein expression in cells analyzed in (A).
(C) Luciferase activity in differentiated Ucp1 luciferase brown adipocytes treated with DMSO (control), forskolin, or rosiglitazone (0.5 μM) for 5 days (n = 3).
(D) UCP1 protein expression in cells analyzed in (C).
(E) Luciferase activity monitored at days 0, 1, 4, and 7 after the start of rosiglitazone treatment in mice implanted with Ucp1 luciferase immortalized preadipocytes. Saline (n = 4) or rosiglitazone (n = 6; 10 mg/kg) treatment started 6 days postimplantation. Representative mice are shown.
(F) Sequential changes in luciferase activity measured in fat transplants from mice in (E).
(G) Hematoxylin and eosin and immunofluorescent stains for UCP1 or GFP (i.e., tdTomato) in transplants from mice treated with rosiglitazone. Merged UCP1 and GFP image counterstained with DAPI. The scale bar represents 50 μm. H&E, hematoxylin and eosin staining.
∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < Data are expressed as mean ± SEM.
Cell Reports 2014 9, DOI: ( /j.celrep )
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5. Figure 3
Small-Molecule Screen to Identify Regulators of UCP1 Expression
(A) Screen scheme. Ucp1 luciferase brown preadipocytes were differentiated in 96-well plates and treated at day 8 with compounds for 16 hr (n = 3).
(B) Screen performance. Mean luciferase activity of compounds (blue circles) plotted relative to the value of DMSO-treated Ucp1 luciferase adipocytes (100%; green circles). Rosiglitazone served as positive control (red circles). Ucp1 luciferase preadipocytes (purple circles) were used to determine background and induction of signal upon differentiation. Hits selected for evaluation (blue circles, red outline) are numbered according to the scheme used in validation experiments.
(C) Luciferase activity in differentiated Ucp1 luciferase brown adipocytes treated with compounds for 5 days. Rosiglitazone (0.5 μM) and CL-316,243 (10 nM) served as positive controls (n = 3). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < versus control. Data are expressed as mean + SD.
(D) UCP1 protein expression in cells from (C).
Cell Reports 2014 9, DOI: ( /j.celrep )
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6. Figure 4 Effect of WWL113 on UCP1 Expression and Cellular Respiration
(A) UCP1 protein expression in differentiated Ucp1 luciferase brown adipocytes treated with WWL113 for 5 days. Rosiglitazone (0.5 μM) served as positive control.
(B) Expression of thermogenic genes in differentiated Ucp1 luciferase brown adipocytes treated with WWL113 (10 μM) for 5 days. Rosiglitazone (0.5 μM) served as positive control (n = 3). ∗p < 0.05; ∗∗∗p < versus control.
(C) Ucp1 mRNA expression in primary differentiated brown adipocytes treated with WWL113 (10 μM) or rosiglitazone (5 μM) for 24 or 48 hr (n = 3). ∗∗∗p < versus control.
(D) Ucp1 mRNA expression in primary differentiated brown adipocytes treated with WWL113 (10 μM) or rosiglitazone (5 μM) for 48 hr. Norepinephrine (0.1 μM) was added 8 hr prior to harvest (n = 3). ∗∗p < 0.01; ∗∗∗p < versus control.
(E) Total and uncoupled (oligomycin-insensitive) respiration of differentiated brown adipocytes (5 × 105 cells/sample) treated with WWL113 (10 μM) in the presence or absence of norepinephrine (0.1 μM; n = 3 to 4). ∗p < 0.05 versus control.
(F) Ucp1 mRNA expression in differentiated brown adipocytes treated with vehicle or WWL113 (10 μM) for 24 hr with or without 30 min pretreatment with a PPARα-selective antagonist (GW6471; 3 μM) and/or a PPARγ-selective antagonist (GW9662; 10 μM; n = 3). ∗∗∗p < versus control. ###p < versus WWL113-treated cells.
(G) Ucp1 mRNA expression in differentiated brown adipocytes treated with vehicle, WWL113 (10 μM), a PPARα-selective agonist (GW9578), or the combination for 24 hr (n = 4). ∗p < 0.05; ∗∗∗p <
Data are expressed as mean + SD.
Cell Reports 2014 9, DOI: ( /j.celrep )
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7. Figure 5 WWL113 Increases UCP1 Expression in Mice
(A) Luciferase activity in Ucp1 luciferase reporter mice treated daily with WWL113 (50 mg/kg) or vehicle for 5 days (n = 5). Representative mice are shown.
(B) Quantification of luciferase signal in interscapular BAT of mice treated as in (A). Values normalized to protein content and shown as fold change relative to vehicle.
(C) Expression of thermogenic genes in interscapular BAT of C57BL/6 mice treated daily with WWL113 (50 mg/kg) or vehicle for 5 days (n = 5). ∗p < 0.05; ∗∗p < 0.01
(D) UCP1 protein expression in interscapular BAT of C57BL/6 mice analyzed in (C).
(E) VO2 of wild-type mice treated daily with WWL113 (50 mg/kg) or vehicle for 7 days (n = 6). CL-316,243 (1 mg/kg) was injected to examine the response to adrenergic stimulation. ∗∗p < N.S., not significant.
(F) Locomotor activity of mice in (E).
(G) Food intake of mice in (E).
(H) Heart rate of mice in (E).
Data are expressed as means ± SEM.
Cell Reports 2014 9, DOI: ( /j.celrep )
Copyright © 2014 The Authors Terms and Conditions